关键词: 纹带棒杆菌, 生物膜, 纹带棒杆菌毒力, 菌毛组装蛋白D, 多克隆抗体

Abstract:

Objective To prepare the polyclonal antibody against pilus assembly protein（SpaD）， a biofilm formation related protein of Corynebacterium striatum（C.striatum）， and to evaluate its inhibitory effect on biofilm formation by strong biofilm-producing strains of C.striatum along with its potential application value. Methods A total of 117 strains of C.striatum isolated from clinical specimens of hospitalized patients at Affiliated Hospital of Inner Mongolia Medical University from 2011 to 2021 were selected as the study subjects. The biofilm formation ability of each strain was detected by crystal violet staining assay. Real-time fluorescence quantitative PCR（RT-qPCR） method was used to examine the distribution of SpaD proteinencoding gene spaD in strong biofilm-producing strains of C.striatum. The strong biofilm-producing strains of C.striatum were divided into control group， and proteinase K group， and cystol violet staning method was used to detect the inhibitory effect of proteinaskon the biofilm formation abilitys of the strong biofilm-producing strains of C.striatum.The recombinant SpaD protein was constructed using protein recombination technology and the was divided into control group and 5 and 10 mg·L^-1 SpaD recombinant proterin groups， and cystol violet staning method was used to detect the inhibitory effect of SpaD recombinant protein. The rabbit anti-SpaD polyclonal antibodies were subsequently obtained through animal immunization， the experiment was divided into control group and 1∶400， 1∶200， and 1∶100 SpaD polyclonal antibody groups， the inhibitory effect of SpaD polyclonal antibodies on biofilm and crystal violet staining method was used to detect abilities of strong biofilm-producing strains of C.striatum. Results The Crystal violet staining results revealed that strong， moderate， and weak biofilm-producing strains accounted for 47.9% （56/117）， 29.0% （34/117）， and 23.1% （27/117）， respectively. The RT-qPCR results showed that all the strong biofilm-producing strains carried the spaD gene. Compared with control group， the biofilm formation abilities of 13 strong biofilm-producing C.striatum strains in both 5 and 10 mg·L^-1 SpaD recombinant protein groups were significantly decreased （P<0.05）. Compared with control group， the biofilm formation abilities in 61.5% （8/13） and 7.7% （1/13） of strong biofilm-producing C.striatum strains in 1∶100 and 1∶200 SpaD polyclonal antibody groups were decreased， respectively （P<0.05）. Conclusions The spaD gene is highly expressed in strong biofilm-producing clinical strains of C.striatum. Anti-SpaD polyclonal antibodies significantly inhibits biofilm formation in these clinical isolates， demonstrating a inhibitory effect as a manner of concentration-dependent. SpaD can be a promising novel target for therapeutic intervention against biofilm-producing C.striatum infections.

Key words: Corynebacterium striatum, Biofilm formation, Virulence of Corynebacterium striatum, Pilus assembly protein D, Polyclonal antibodies